Agents for imaging soluble a-beta

ABSTRACT

Provided herein are agents that bind to soluble beta-amyloid. Also provided are in vivo and in vitro methods for detecting soluble beta-amyloid in a sample that may include brain tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending U.S. patentapplication Ser. No.10/747,715, filed Dec. 26, 2003.

FIELD OF THE INVENTION

The present disclosure relates to methods for the detection of solublebeta-amyloid and the measurement of its local concentration in a sample.In some embodiments, the sample may the brain of a subject and themeasurement may occur without invasive procedures. The presentdisclosure also relates to agents that are useful for detecting solubleBeta-Amyloid in either in vitro or in vivo.

BACKGROUND

The main histopathological characteristic of Alzheimer's disease (“AD”)is the presence of neuritic plaques and tangles combined with associatedinflammation in the brain. It is known that plaques are composed mainlyof deposited (or insoluble in aqueous solution) fibrillar forms of thebeta-amyloid (“A-beta”) peptide. The formation of fully fibrillaraggregated A-beta peptide is a complex process that is initiated by thecleavage of the amyloid precursor protein (“APP”). After cleavage ofAPP, the monomeric form of A-beta may associate with other monomers,presumably through hydrophobic interactions or domain swapping, to formdimers, trimers and higher-order oligomers. Oligomers of A-beta mayfurther associate to form protofibrils and eventual fibrils, which isthe main constituent of neuritic plaques. Soluble oligomers (soluble inaqueous buffer) of A-beta may contribute significantly to neuronaldysfunction. In fact, animal models suggest that simply lowering theamount of soluble A-beta peptide, without affecting the levels of A-betain plaques, may be sufficient to improve cognitive function.

Presently, the only definitive method of AD diagnosis is postmortemexamination of brain for the presence of plaques and tangles. Theantemortem diagnosis of AD is difficult, especially during the earlystages, as AD symptoms are shared among a spectrum of other dementias.Currently, AD diagnosis is achieved using simple cognitive testsdesigned to test a patient's mental capacity such as, for example, theADAS-cog (Alzheimer's disease assessment scale—cognitive subscale) orMMSE (Mini-mental state examination). The subjective nature and inherentpatient variability is a major shortcoming of diagnosing AD by suchcognitive means. The fact that AD cannot be accurately diagnosed earlycreates a formidable challenge for pharmaceutical companies that aim totest anti-A-beta drugs as therapy to slow or halt AD pathogenesis.Furthermore, even if AD could be detected early and patients could betreated with A-beta lowering compounds, there is currently no way toknow if the therapy is clinically efficacious. Therefore, a significantneed exists to develop methods of measuring the soluble A-beta peptidelevels locally in the brain.

Diagnosing AD by directly measuring levels of beta-amyloidnon-invasively has been attempted by the targeted imaging of senileplaques. This approach fails as a specific measure of soluble A-betapeptide because current A-beta targeted imaging agents are directed atinsoluble aggregates that are characteristic of A-beta fibrillardeposits in the brain. Small molecules that specifically bind toinsoluble A-beta deposits include, for example, Congo red, Chrysamine G,methoxy-X04, TZDM, [¹¹C]6, IMSB, Thioflavin(e) S and T, TZDM, 1-BTA,benzathiozole derivatives, [¹²⁵I]3, BSB, IMSB,styrylbenzene-derivatives, IBOX, benzoxazole derivatives, IMPY, pyridinederivatives, DDNP, FDDNP, FENE, dialkylaminonaphthyl derivatives, andcertain benzofuran derivatives (see, e.g., U.S. Pat. Nos. 6,133,259;6,168,776; and 6,114,175).

Certain nucleic acid sequences have been shown to bind to insolublesenile plaques of A-beta, including mRNA for furin and amyloid precursorprotein (“APP”).

Peptides also have been developed as imaging agents for insolubledeposits of A-beta and senile plaques. The sequence specific peptidesthat have been labeled for the purpose of imaging insoluble A-betaincludes the labeled A-beta peptide itself, putrescine-gadolinium-A-betapeptide, radiolabeled A-beta, [¹¹¹In]A-beta, [¹²⁵I]A-beta, A-betalabeled with gamma emitting radioisotopes, A-beta-DTPA derivatives,radiolabeled putrescine, and KVLFF-based ligands.

Inhibitors of aggregated A-beta have been suggested to disrupt theformation of these aggregates by interacting with soluble or insolublefibrils of A-beta. Examples of inhibitors or anti-aggregation agentsinclude peptides of A-beta, KVLFF-based ligands, small molecular weightcompounds, carbon nanostructures, rifamycin, IDOX, acridone, benzofuran,and apomorphine. Agents have also been identified that promote A-betaaggregation (e.g., agents such as A-beta42, proteins, metals, smallmolecular weight compounds, and lipids).

Targeted imaging of plaques may not provide early diagnosis, as largeplaque burden is mostly associated with mid-to-late stage disease.Moreover, it has not been shown that current anti-A-beta therapies willaffect fibrillar deposits appreciably to detect by imaging techniques atclinically relevant time points.

In vitro measures of A-beta may be specific for soluble A-beta in thecerebral spinal fluid, but lacks the necessary selectivity for localA-beta in the brain that is necessary for direct, accurate assessment ofbrain levels of soluble A-beta species. To date, the targetednon-invasive measurement and imaging of soluble A-beta peptide speciesthat exist in the central nervous system have not been addressed.

SUMMARY

This disclosure relates a compound having the following Formula I

wherein X is selected from a group that comprises at least one ofoxygen, nitrogen and sulfur; R¹ is selected from the group consisting ofsubstituted or unsubstituted alkyl hydroxy, amide, urea, and urethane;and R² is a hydrocarbon radical selected from the group consisting of aC₁-C₃₂ substituted or unsubstituted branched or straight chain alkyl,cycloaliphatic, aryl and heteroaryl, including five membered rings, sixmember rings, and fused systems thereof.

In another aspect, an imaging agent is described, comprising thecompound described in Formula I and a label. In yet another aspect,methods of detecting at least one of A-beta species and amyloidogenicpeptides comprising the steps of providing a sample suspected ofcomprising A-beta species or amyloidogenic peptides, applying an imagingagent comprising a compound described in Formula I, and detecting anamount of imaging agent bound to the at least one of A-beta species andamyloidogenic peptides.

In another aspect, methods of assessing an amyloid -related diseasecomprising the steps of administering to a subject an imaging agentcomprising a compound as described in Formula I and detecting theimaging agent bound to at least one of A-beta species and amyloidogenicpeptides.

In yet another aspect, methods of non-invasively assessing thetherapeutic efficacy of therapies in a subject are described whichinclude the steps of administering to a subject a first dose of acomposition described in Formula I, and non-invasively obtaining abaseline measurement of the imaging agent within the subject,administering to the subject a therapy to be evaluated, administering tothe subject a second dose of said composition, non-invasively obtaininga second measurement of the imaging agent within the subject, andcomparing the two or more measurements separated in time, wherein anincrease or decrease in the amount of the imaging agent presentindicates the efficacy of the therapy.

FIGURES

FIG. 1 depicts SPA saturation binding curve of ³H-49b with solubleA-beta oligomers and fibrils.

FIG. 2 shows SPA self-competition assay between labeled and unlabeled49b on soluble A-beta oligomers.

FIG. 3 compares binding of tritiated 49b with other tritiated probes inbinding to soluble A-beta oligomers. Tritiated 49b was compared withnon-related molecules such as tritiated cimetide, caffeine, and AZT.

FIG. 4 compares binding of tritiated with related benzofuran analogs 37band 66b.

FIG. 5 depicts AFM images of oligomers (Panel A) and fibrils (Panel D)in solution; and oligomers bound to PVT-Streptavidin SPA beads (Panel B)and fibrils (Panels E and F) bound to PVT-Streptavidin SPA beads. Thesurface of SPA beads alone (without oligomers or fibrils) is shown inPanel C. All images, except the image in Panel F, are 1 um×1 um images.The image shown in Panel E corresponds to the area highlighted by asquare in Panel F. Panel E is a topographical image, while all the restare phase images.

FIG. 6 shows binding of the 49b to synthetic A-beta oligomers in thecontext of a rat brain slice (Panels A and B), fibrils (Panels C and D).6E10 immunostaining of the sections were in the top panels (A-D). InPanels A and C, Thioflavin T was used to check for presence of crossedbeta-sheets in the synthetic preparations (bottom). In Panels B and D,the slides were exposed to 49b (bottom)

FIG. 7 demonstrates co-localization of 6E10 antibody with the benzofuranderivative, 49b. Panel A and B show serial staining of brain samplederived from 24-month old PDAPP transgenic mice. Panel C shows the sameserial staining in a control animal, which is a 3 month old PDAPPtransgenic mice (Panel C). The scale bar in C is 50 microns.

FIG. 8 shows 49b staining in 24-month old PDAPP co-localized withThioflavin S-positive staining from the mouse hippocampal sample.Because 49b and Thioflavin S have similar spectral properties,co-localization was demonstrated by staining the sections first with49b, then washing the sections to remove the first signal, followed bystaining with Thioflavin S. Sections shown in Panels A and B were takenfrom two different PDAPP mice. The scale bar in Panel B corresponds to50 microns.

FIG. 9 shows 49b and Thioflavin S binding on 24-month old PDAPP brainsections in which soluble oligomers were removed using carbonatepre-treatment. The samples shown in Panel A and Panel C were pre-treatedwith PBS, while the samples shown in Panel B and Panel D werepre-treated with carbonate prior to incubation with 49b (Panels A andB), and Thioflavin S (Panels C and D). The scale bar is 50 microns.

DETAILED DESCRIPTION

The following detailed description is exemplary and not intended tolimit the invention of the application and uses of the invention.Furthermore, there is no intention to be limited by any theory presentedin the preceding background of the invention of the following detaileddescription of the drawings.

To more clearly and concisely describe and point out the subject matterof the claimed invention, the following definitions are provided forspecific terms that are used in the following description and the claimsappended hereto.

As used herein, the term “antibody” refers to an immunoglobulin thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule.Antibodies useful in present invention may be monoclonal or polyclonaland may be prepared by art-recognized techniques such as immunization ofa host and collection of sera (polyclonal) or by preparing continuoushybrid cell lines and collecting the secreted protein (monoclonal), orby cloning and expressing nucleotide sequences or mutagenized versionsthereof coding at least for the amino acid sequences required forspecific binding of natural antibodies. Antibodies of the variousclasses and isotypes (e.g., IgA, IgD, IgE, IgG1, IgG2a, IgG2b, IgG3, andIgM) may also include a complete immunoglobulin or functional fragments.In addition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments may be used where appropriate so long as an appropriatebinding affinity for a particular molecule is maintained.

As used herein, the term “A-beta species” generally refers to thevarious forms of A-beta-derived polypeptide identified by SEQ ID: 1. Ingeneral, A-beta may comprise amyloid polypeptides of varying length,various aggregation states, and/or solubility. The term “A-beta species”is intended to encompass A-beta species of varying polypeptide lengths.Thus, A-beta species may include various forms of A-beta amino acidresidues 1 through 43 of the full length A-beta peptide shown in SEQ. IDNO:1. Alternatively, the A-beta species may consist essentially of:residues 1-42 of the full length A-beta peptide, residues 1 through 40of the full length A-beta peptide, residues 1-39 of the full lengthA-beta peptide, residues 1-38 of the full length A-beta peptide,residues 3-40 of the full length A-beta peptide, residues 3-42 of thefull length A-beta peptide, residues 11-40 of the full length A-betapeptide, residues 11-42 of the full length A-beta peptide, residues 17-40 of the full length A-beta peptide, and residues 17-42 of the fulllength A-beta peptide.

The general term A-beta species also encompasses various forms of A-betain several aggregation states (e.g., monomeric, soluble oligomers, orinsoluble oligomeric). Because a variety of factors may affect whichspecies of A-beta is found in solution, the aggregation state of theA-beta species may be selected according to the user's purposes byaltering the A-beta polypeptide length, increasing or decreasing theconcentration of A-beta present in a given aliquot of A-beta species,increasing or decreasing the temperature, pH, salt levels and metalcontent (e.g., Zn²⁺, Cu²⁺, etc.) of the given aliquot of A-beta species.

Various forms of soluble or insoluble A-beta species (regardless of thelength of the polypeptide or the association state) may be derived froma variety of mammalian tissue sources, including but not limited to,brain tissue, cerebrospinal fluid, or blood serum. Alternatively, theA-beta species may be synthesized using art-recognized techniques suchas protein expression systems or peptide synthesizers.

“Amyloidogenic peptides” as used herein refer to peptides or proteinsthat have underwent or have the propensity to undergo an amyloidogenicprocess to form aggregates called amyloid, which have a secondarystructure of cross-beta sheets, are birefringent under polarized light,stain with the histological stain Congo red, and are fibrillar innature.

As used herein the term “complexed” generally refers to the aggregationstate of A-beta species in solution (e.g., monomeric or multimericaggregates of the A-beta polypeptide). Thus, the terms “complex” and“oligomer,” as used herein, refer to the polypeptides in the associatedor bound state. And, the term “monomer” refers to a single peptide chainof A-beta.

As used herein, the terms “fibrils” and “fibrillar” generally refer toA-beta preparations with largely beta-sheet content that are insolubleaggregates. Fibrils bind Congo Red and Thioflavin T dyes and cause thesedyes to produce fluorescence signal. Fibril preparations preferentiallycomprise substantially fibrillar A-beta, but they may also compriseunsubstantial amounts of globular aggregates.

As used herein, the term “soluble oligomers” generally refers tocomplexed A-beta polypeptides short chain of monomers derived from theA-beta polypeptide, preferentially less than 25 monomers in length, butmay be 100 monomers in length.

As used herein, the term “signal generator” encompasses a substance thatis capable of being detected by an imaging modality (e.g., opticaldetection or radiography) in the course of the disclosed methods.Examples of signal generators include, but are not limited to,fluorophores (e.g., cyanine dyes), radioisotopes, and paramagnetic ions.In some instances, the soluble A-beta binder.

As used herein, the term “small molecule” refers to an organic molecule,either naturally occurring or synthetic, that has a molecular weightless than about 5000 daltons, and preferentially in the range of about200 to about 2000 daltons.

As used herein with regard to the A-beta species, the term “soluble”generally refers to nonaggregated A-beta peptides that are relativelystable and exhibit structural and functional characteristics that aredistinct from the fibrillar amyloidogenic form of A-beta. In general theaggregation status of A-beta peptides may be broken into threecategories: (1) micelles; (2) protofibrils; and (3) fibrils. Theaggregation state of A-beta species may be determined using thetechniques set out in Goldsbury et. al, J Struct Biol.; June;130(2-3):352-62, (2000), in which samples are classified by the amountof β strands in undisturbed solution (pH 7.4 at 37° C.) by circulardichroism. Under these conditions (1) micelles demonstrate 0% β strands;(2) protofibrils demonstrate about 76% β strands; and (3) fibrilsdemonstrate 100% β strands. Soluble A-beta species for the assays of theinvention contain only insubstantial amounts of protofibrils andfibrils.

As used herein, the term “preferentially binds” refers to the specificrecognition of one of two different molecules for the other compared tosubstantially less recognition of other molecules. Thus, an agent thatspecifically binds a target molecule demonstrates affinities at leastfive-fold, and preferentially 10-fold to 100-fold affinities greaterthan non-binders. Examples of specific binding include antibody-antigeninteractions, enzyme-substrate interactions, polynucleotideinteractions, and so forth.

An agent exhibits “specific binding” if it reacts or associates morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular target (e.g., cells or substances) than itdoes with alternative targets. The specific binders for a particularA-beta species associates with one or more A-beta species with highaffinity for example, an affinity constant of at least 10⁷ M⁻¹,preferably between 10⁸ M⁻¹ and 10¹⁰ M⁻¹, or about 10⁹ M⁻¹.

As used herein, the term “species-specific binder” refers to any binderthat preferentially attaches to one particular species of A-beta (e.g.,soluble A-beta) relative to other species of A-beta (e.g., fibrillarA-beta).

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,so forth used in the specification and claims are to be understood asbeing modified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Imaging Methods

The present disclosure relates to methods of non-invasively assessinglevels of soluble A-beta. Among other uses, the methods disclosed hereinmay be employed to diagnose or monitor amyloid-related diseases,including AD. In some embodiments, the disclosed methods may be used toqualitatively or quantitatively determine soluble A-beta levels in vivoor ex vivo. In other embodiments, these methods may also be used todetermine the efficacy of related therapies used for amyloid-relateddiseases.

The imaging modality may include positron emission tomography (“PET”),optical, single photon emission computed tomography (“SPECT”), magneticresonance imaging (“MRI”), ultrasound, computed tomography (“CT”),depending on the label used, the modality available to medical personneland the medical needs of the subject. Equipment and methods for theforegoing imaging modulations are known to those skilled in the art.

To assess the soluble A-beta levels, a labeled diagnostic imaging agentis delivered to a subject. Typically, the subject is a mammal and may behuman. The labeled imaging agent contains at least a chemical entitythat binds to soluble A-beta and a chemical entity (e.g., a signalgenerator) that emits a signal detectable by an imaging modality that iscompatible with the signal generator. The labeled imaging agent may bedelivered to a subject by a medically appropriate means. After allowinga clearance time according to the label chosen, the amount of imagingagent bound to soluble A-beta is determined by measuring the emittedsignal using an imaging modality. The visual and quantitative analysesof the resulting images provide an accurate assessment of the global andlocal levels of soluble A-beta in the brain.

The present disclosure relates to a method of non-invasively assessinglevels of soluble A-beta to diagnose amyloid-related diseases, includingAlzheimer's disease. This method qualitatively and quantitativelydetermines soluble A-beta levels in vivo. This method can also be usedto determine the efficacy of related therapies used for amyloid-relateddiseases. To assess the soluble A-beta levels, a labeled diagnosticimaging agent is delivered to a subject. Typically, the subject is amammal and can be human. The labeled imaging agent contains at least achemical entity that binds to soluble A-beta and a chemical entity thatemits a signal detectable by an imaging modality. The labeled imagingagent is delivered to a subject by a medically appropriate means. Afterallowing a clearance time according to the label chosen, the amount ofimaging agent bound to soluble A-beta is determined by noninvasivelymeasuring the emitted signal using an imaging modality. The visual andquantitative analyses of the resulting images provide an accurateassessment of the global and local levels of soluble A-beta in thebrain.

The present disclosure also relates to a method of labeling anddetecting A-beta species and amyloidogenic peptides as well asquantitatively measuring the amount of A-beta species and amyloidogenicpeptides in vitro, ex vivo, and in situ. The agent that binds to A-betaspecies and amyloidogenic peptides is detected by the agent's emittedsignal, such as emitted radiation, fluorescence emission, and opticalproperties of the agent. A-beta species and amyloidogenic peptides fromat least cell culture, post-mortem human tissue, animal models ofdisease, and synthetic and recombinant sources are typically exposed toexcess agent for a period of incubation. Agent that is nonspecificallybound or free in the incubation solution is blocked or washed away toleave agent specifically bound to A-beta species and amyloidogenicpeptides that is detectable with common microscopic, wide field imaging,radiometric, fluorescent, optical, and analytical techniques. Thedetectable signal may be converted to numerical values to quantify theamount of targeted A-beta species and amyloidogenic peptides.

The chemical entity of the imaging agent that binds to soluble A-betacan bind to monomers, dimers, trimers, and/or oligomers comprised of alarger number of A-beta peptides up to 24 A-beta peptides. Morespecifically, the soluble A-beta species to which the imaging agent canbind include monomers, dimers, trimers, and oligomers of A-beta 1-38,A-beta 1-39, A-beta 1-40, A-beta 1-41, A-beta 1-42, A-beta 1-43 or anycombination thereof. The A-beta peptide in soluble monomer or oligomerforms can be derived ex vivo, by recombinant means, or synthetically.The soluble A-beta includes monomeric and low oligomeric A-beta that issoluble in an aqueous solution. In some embodiments, the soluble A-betais of a type that remains in the supernatant of aqueous solution aftercentrifugation at 15000 times gravity. In some embodiments, the solubleA-beta includes A-beta monomers and its aggregates that do not exhibitgreen birefringence when stained by Congo red.

The imaging agent that binds to soluble A-beta or otherwise reports onthe presence of soluble A-beta can be derived from a natural source orbe man made and be a small molecule, peptide, protein, enzyme, nucleicacid, nucleic acid sequence, dendrimer, polymer, antibody or antibodyfragment.

As well known in the art, such compounds may be found in compoundlibraries, combinatorial libraries, natural products libraries, andother similar sources, and may further be obtained by chemicalmodification of compounds found in those libraries, such as by a processof medicinal chemistry as understood by those skilled in the art, whichcan be used to produce compounds having desired pharmacologicalproperties.

In one embodiment, certain compounds and their derivatives are useful asimaging agents that bind to soluble A-beta. The compositions describedherein exhibit nanomolar affinity to soluble A-beta, as determined byfluorescence binding assays. Suitable compounds and derivatives includethose represented by the following Formula I:

wherein X is selected from a group that comprises at least one ofoxygen, nitrogen, or sulfur; R¹ is a hydrocarbon radical; and R² is ahydrocarbon radical or halogen. Preferably, R² is a hydrocarbon radical.By halogen is meant to include any of the entities known by those ofordinary skill in the art to be halogens, including, but not limited tobromine, fluorine, chlorine, and iodine. By hydrocarbon radical is meantany hydrocarbon radical, including substituted and unsubstituted,saturated or unsaturated, branched, straight chain alkyl, aryl,heteroaryl, cycloalphatic radicals, or any combination of the above. R¹and R² may further be substituted with one or more heteroatom of oxygen,nitrogen, sulfur, or halogen, such as chlorine, bromine, or fluorine. Inone embodiment, R¹ is C₁-C₁₀ alkyl hydroxy, an amide group, a ureagroup, or a urethane group and R² is a C₁-C₃₂ branched or straight chainalkyl, substituted or unsubstituted; a substituted or unsubstituted arylgroup; a substituted or unsubstituted cycloaliphatic group.

The term aryl and heteroaryl as used herein is intended to include, butnot be limited to, five membered rings, six membered rings and fusedring systems thereof. The term “alkyl” as used in the variousembodiments of the present invention is intended to designate bothlinear alkyl, branched alkyl, aralkyl, cycloalkyl, bicycloalkyl,tricycloalkyl and polycycloalkyl radicals containing carbon and hydrogenatoms, and optionally containing atoms in addition to carbon andhydrogen, for example atoms selected from Groups 15, 16 and 17 of thePeriodic Table, including but not limited to oxygen, sulfur, nitrogen,fluorine, bromine and chlorine. The term “alkyl” also encompasses thatalkyl portion of alkoxide groups. In various embodiments normal andbranched alkyl radicals include as illustrative non-limiting examplesC₁-C₃₂ alkyl optionally substituted with one or more groups selectedfrom C₁-C₁₂ alkyl, C₃-C₁₅ cycloalkyl or aryl; and C₃-C₁₅ cycloalkyloptionally substituted with one or more groups selected from C₁-C₃₂alkyl. Some particular illustrative examples comprise methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, tertiary-butyl, pentyl,neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl.Some illustrative non-limiting examples of cycloalkyl and bicycloalkylradicals include cyclobutyl, cyclopentyl, cyclohexyl, methylcyclohexyl,cycloheptyl, bicycloheptyl, and adamantyl. In various embodimentsaralkyl radicals are those containing from about 7 to about 14 carbonatoms; these include, but are not limited to, benzyl, phenylbutyl,phenylpropyl, and phenylethyl. In various embodiments aryl radicals usedin the various embodiments of the present invention are thosesubstituted or unsubstituted aryl radicals or fused aromatic radicalscontaining from 6 to 18 carbon atoms. Some illustrative non-limitingexamples of these aryl radicals include C₆-C₁₅ aryl optionallysubstituted with one or more groups selected from C₁-C₁₂ alkyl, C₃-C₁₅cycloalkyl, or aryl. Some particular illustrative examples of arylradicals comprise substituted or unsubstituted phenyl, biphenyl, toluyl,and naphthyl.

In yet another embodiment of the present invention, R¹ is alkylhydroxyl, an amide group, a urea group or a urethane group such as thoseof the following formula:

wherein Z is NH or S, or R¹ has the following structure

wherein Y is H or O, n is equal to the integer 1 or 2, R³ is O or NH,and R⁴ is an acyl, substituted or unsubstituted aryl, ureas, orurethanes. Further, nonlimiting examples of R² include those shown inTable 1 below.

TABLE 1

1a

2a

3a

4a

5a

6a

7a

8a

9a

10a

11a

12a

13a

14a

15a

16a

17a

18a

20a

19a

21a

22a

23a

24a

25a

26a

27a

28a

29a

30a

31a

32a

33a

34a

35a

36a

37a

38a

39a

40a

41a

42a

43a

44a

45a

46a

47a

48a

49a

50a

51a

52a

53a

In yet another embodiment, useful imaging agents include benzofuranderivatives such as those of the following Formula II:

wherein R¹ and R² are as described above. More particularly, certainbenzofuran derivatives having a formula as described in Table 2 areuseful as imaging agents for A-beta.

TABLE 2

1b

2b

3b

4b

5b

6b

7b

8b

9b

10b

11b

12b

13b

14b

15b

16b

17b

18b

19b

20b

21b

22b

23b

24b

25b

26b

27b

28b

29b

30b

31b

32b

33b

34b

35b

36b

37b

38b

39b

40b

41b

42b

43b

44b

45b

46b

47b

48b

49b

50b

51b

52b

53b

54b

55b

56b

57b

58b

59b

60b

61b

62b

63b

64b

65b

66bwherein Z is NH, O, S; Ar is substituted phenyl, pyridinyl, thiophenyl,furanyl, as in the examples above. As is known in the art, “Et” is ethyland “Me” is methyl.

Substituted 2-phenyl-3-alkyl benzofurans similar to those shown inFormula II have been synthesized by the Lewis acid—mediated condensationof benzyl with phenols and aryl ethers, Palladium—catalyzedcross-coupling of benzyl ketones and α,β-unsaturated carbonyl andphenolic compounds with o-dibromobenzenes, and McMurry-type reductivecyclization of dicarbonyl compounds catalyzed by low-valent Ti. Also,2,3-diphenyl benzofurans have been prepared by the cyclodehydration ofα-aryloxydeoxybenzoins catalyzed by phosphoric acid.2-Aryl-3-allylbenzofurans have been synthesized by the Pd-catalyzedcyclization of 2-alkynylphenols with allyl carbonates. Pd-catalyzedcyclizations are also the preferred method for the synthesis of avariety of 2-alkylbenzofurans and 2-arylbenzofurans.

In yet another embodiment, useful imaging agents include benzofuranderivatives such as those of the following Formula III:

wherein R¹ is as described above, and R3 is an aryl, alkyl substitutedaryl, particularly fluoroethyl substituted alkyl, substituted heteroarylmoieties such as furans, thiophenes, indoles, imidazoles, oxazoles,thiazoles, oxadiazoles, thiadiazoles and triazoles.

More particularly, certain benzofuran derivatives having a formula asdescribed in Table 3 are useful as imaging agents for A-beta.

TABLE 3

1c

2c

3c

4c

5c

6c

7c

8c

9c

10c

11c

12c

13c

14c

15c

49bAntibodies Against Soluble A-beta

Anti-A-Beta antibodies may be prepared against a suitable antigen orhapten comprising the desired target epitope, such as the junctionregion of the 1-42 A-beta oligomer consisting of amino acid residues13-26. An alternative antigen or hapten may include the carboxy terminusconsisting essentially of amino acid residues 33-42 of A-beta. Onesuitable antibody to soluble A-beta is disclosed in Kayed, et al.,Science, Vol. 300, p. 486, Apr. 18, 2003 (incorporated by referenceherein). The target haptens (e.g., synthetic peptides comprising anA-beta epitope) may also be prepared by conventional solid phasetechniques, coupled to a suitable immunogen, and used to prepareantisera or monoclonal antibodies. Suitable peptide haptens typicallywill typically comprise at least five contiguous residues within A-betaand may include more than six residues. Synthetic polypeptide haptensmay be produced by the Merrifield solid-phase synthesis technique inwhich amino acids are sequentially added to a growing chain (Merrifield(1963) J. Am. Chem. Soc. 85:2149-2156). Suitable antibodies may include,for example, those of antibodies described in U.S. Pat. Nos. 5,811,310;5,750,349; and 5,231,000, R1282, 21F12, 3D6, FCA3542, and othermonoclonal and polyclonal antibodies for A-beta 1-40, and/or A-beta 1-42that demonstrate the ability to preferentially or specifically bind tosoluble A-beta.

Activatable Agents

Imaging agents have been developed that may report on the specificpresence of a target molecule without binding to that molecule. In suchinstances the imaging agents are considered “activatable” because theirsignal is activated or unactivated based on the presence of a specifictarget molecule. Examples of such agents have been used for MRI andoptical imaging.

Signal Generators

In some embodiments, the soluble A-beta binding agent includes anintrinsic signal generator (e.g., a radiolabel or a fluorescent moiety)and requires no additional components to be observed using a selectedmodality (e.g., optical detection or radiography). In embodiments wherethe soluble A-beta binding agent does not include an intrinsic signalgenerator, the imaging agent may be supplemented with one or more signalgenerators capable of being detected by a particular imaging modality.

Examples of signal generators include, but are not limited to,fluorophores (e.g., cyanine dyes), radioisotopes, and paramagnetic ions.Suitable radioisotopes may include ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ¹²³I,¹²⁵I, ¹³¹I, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁹Fe, ⁷⁵Se, and ¹⁵²Eu. Isotopes ofhalogens (such as chlorine, fluorine, bromine and iodine), and metalsincluding technetium, yttrium, rhenium, and indium are also usefullabels. Typical examples of metallic ions that may be used as signalgenerators include ^(99m)Tc, ¹²³I, ¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, ¹²⁵I,⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹TI.

For use with the present disclosure, radiolabels may be prepared usingstandard radiolabeling procedures well known to those skilled in theart. For example, the labeling will be accomplished by incorporation ofone of the above-listed labels into one or both of the R¹ or R² groupsinto the benzofuran derivative shown as Formula I, Formula II, orFormula III.

The disclosed compounds may be radiolabeled either directly byincorporating the radiolabel directly into the compounds or indirectlyby incorporating the radiolabel into the compounds through a chelatingagent, where the chelating agent has been incorporated into thecompounds. Such radiolabeling should also be reasonably stable, bothchemically and metabolically, applying recognized standards in the art.Also, although the label may be incorporated in a variety of fashionswith a variety of different radioisotopes, such radiolabeling should becarried out in a manner such that the high binding affinity andspecificity of the unlabeled binding moiety is not significantlyaffected.

Preferred radioisotopes for in vivo diagnostic imaging by positronemission tomography (“PET”) are ¹¹C, ¹⁸F, 123I, and 125I, with ¹⁸F beingthe most preferred. Typically, the labeled atom is introduced to thelabeled compounds at a late stage of the synthesis. This allows formaximum radiochemical yields, and reduces the handling time ofradioactive materials. When dealing with short half-life isotopes, animportant consideration is the time required to conduct syntheticprocedures, and purification methods. Protocols for the synthesis ofradiolabeled compounds are described in Tubis and Wolf, Eds.,“Radiopharmacy”, Wiley-Interscience, New York (1976); Wolf, Christman,Fowler, Lambrecht, “Synthesis of Radiopharmaceuticals and LabeledCompounds Using Short-Lived Isotopes”, in Radiopharmaceuticals andLabeled Compounds, Vol. 1, p. 345-381 (1973).

Paramagnetic labels may be metal ions are present in the form of metalcomplexes or metal oxide particles. Suitable paramagnetic isotopes mayinclude ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe. The paramagnetic label maybe attached to the binding moiety by several approaches. One approach isdirect attachment of one or more metal chelators to the binding moietyof the imaging agent. Alternatively, the binding portion of the imagingagent may be attached to a paramagnetic metal ion or heavy atomcontaining solid particle, or to an echogenic gas microbubble. A numberof methods may be used to attach imaging agent, which specifically bindsto soluble A-beta, to paramagnetic metal ion, or heavy atom containingsolid particles by one of skill in the art of the surface modificationof solid particles. In general, the imaging agent is attached to acoupling group that reacts with a constituent of the surface of thesolid particle. The coupling groups may be any of a number of silanes,and also include polyphosphonates, polycarboxylates, polyphosphates ormixtures thereof, which react with surface hydroxyl groups on the solidparticle surface, as described, for example, in U.S. patent applicationpublication 2002/0159947 and which may couple with the surface of thesolid particles, as described in U.S. Pat. No. 5,520,904.

The imaging agent itself may be fluorescent or may be tagged with asignal generator (e.g., an optical label that are fluorophores) such asfluorescein, rhodamine, Texas Red, and derivatives thereof. The opticallabel may be chemiluminescent, such as green fluorescent protein,luciferin, dioxetaneor. The soluble-A-beta binder may be linked to theportion of the signal generator using techniques known to those skilledin the art.

Binding Assays

Also provided herein are various methods for determining the ability ofa putative binder to bind an A-beta species comprising the steps of: (a)applying one of the inventive compounds that bind soluble A-beta to asample with at least one A-beta species; (b) applying a putative binderbind soluble A-beta to a the same or a separate sample; and (c)determining whether the putative binder binds to the A-beta species.

In some embodiments, the disclosed methods provide additional controlsteps wherein each of steps (a)-(c) are repeated using a one of theinventive compounds (i.e., a validated compound) in place of theputative binder to provide a positive control. Thus, for the positivecontrol variant, steps (a)-(c) may be repeated using a validated binderin place of the putative binder. Likewise, for the negative controlvariant, steps (a)-(c) may be repeated using a validated nonbinder inplace of the putative binder. For either positive control or negativecontrol variations the additional control steps (a)-(c) may be performedeither in parallel or in tandem with the assay for the putative binder.In all embodiments including one or more control the methods may includethe additional step of determining the relative binding of the putativebinder and the validated binder or the validated non-binder. Thedetermining binding step may entail any art recognized methods ofdetermine binding in a tissue sample, for example optically observingthe binding using fluorescence microscopy or radiographic imaging. Wherethe binder has an inherent ability to general a measurable signal whenpresent in a sample (e.g. fluoresces or generates a radioactive signal),no additional agents are required for the determining binding step. If,however, the binder does not have the ability to generate signal, thedisclosed methods additional steps of applying a signal generator tofacilitate the binding determination step. Such an additional step mayemploy, for example, a labeled antibody that recognizes the binder.

Modes of Administration

The labeled imaging agent may typically be administered to a patient ina composition comprising a pharmaceutical carrier. A pharmaceuticalcarrier may be any compatible, non-toxic substance suitable for deliveryof the labeled or unlabeled A-beta binding agents to the patient,including sterile water, alcohol, fats, waxes, proteins, and inertsolids may be included in the carrier. Pharmaceutically acceptableadjuvants (e.g., buffering agents, dispersing agent) may also beincorporated into the pharmaceutical composition. Carriers may contain asolution of the imaging agent or a cocktail thereof dissolved in anacceptable carrier, preferably an aqueous carrier. A variety of aqueoussterile carriers may be used, e.g., water, buffered water, 0.4% saline,0.3% glycine, or 25% human serum albumin.

The solutions or compositions comprising the soluble A-beta bindingagents are preferably pyrogen-free, sterile, and generally free ofparticulate matter. The solutions or compositions may contain additionalpharmaceutically acceptable substances as necessary to approximatephysiological conditions such as pH adjusting and buffering agents,toxicity adjusting agents, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate.

The concentration of imaging agent in the composition or solutions mayvary as required. Typically, the concentration will be in trace amountsto as much as 5% by body weight of the subject but with vary accordingto the particular imaging modality used. Typically, agent concentrationsare selected primarily based on fluid volumes, and viscosities inaccordance with the particular mode of administration selected.Preferably, the agent concentration is between about 0.1 and about 1nmol, more preferably from about 0.1 nmol to about 0.5 nmol. The imagingagent may be present in several ml of injectable solution, as would bedetermined based on dose, and easily calculated by one of ordinary skillin the art. For example, if the agent is labeled with ¹⁸F, approximately105 pmol of ¹⁸F yields 10 mCi of a radiation dose initially. This amountof radioactivity is typical and considered safe in the current medicalimaging procedures. A typical composition for intravenous infusion maybe made to contain 250 ml of sterile Ringer's solution and up to 100 mg,preferably around 10 mg, of the soluble A-beta imaging agent. Thecomposition containing the imaging agent may be combined with apharmaceutical composition and may be administered subcutaneously,intramuscularly, or intravenously to patients suffering from, or at riskof, amyloid-related conditions such as AD.

In some embodiments, clearance time can be employed to permit theportions of the imaging agent to travel throughout the subject's bodyand bind to any available soluble A-beta while also permitting theunbound imaging agent to be cleared from the body or from the brain tothereby decrease noise resulting from non-bound imaging agent. In caseswhere the imaging agent does not directly bind, but rather reports onthe presence of the A-beta, sufficient time is allowed for a specificinteraction to occur in which the reporter molecule is activated. Theclearance time will vary depending on the label chosen for use and mayrange from 1 minute to 24 hours.

The imaging agent may be delivered and the imaging taken to determinethe amount of soluble A-beta present in the subject's body as anindication of disease or pre-disease states. The levels of solubleA-beta may be indicative of pre-disease conditions and therapies towardremoval of the soluble A-beta or its precursors may prevent or forestallthe onset of an amyloid-related disease, such as AD.

Therapeutic Efficacy

In another aspect, the present methods may be used to determine theefficacy of therapies used in a subject. By using multiple images overtime, the levels of A-beta may be tracked for changes in amount andlocation. This method may aid physicians in determining the amount andfrequency of therapy needed by an individual subject. In thisembodiment, an imaging agent in accordance with the present disclosureis administered and a baseline image is obtained. The therapy to beevaluated is administered to the subject either before or after abaseline images are obtained. After a pre-determined period of time, asecond administration of an imaging agent in accordance with theirdisclosure is given. A second or more images are obtained. Byqualitatively and quantitatively comparing the baseline and the secondimage, the effectiveness of the therapy being evaluated may bedetermined based on a decrease or increase of the signal intensity ofthe second image or additional images.

EXAMPLES

The following non-limiting Examples are shown and describe variousembodiments of the present invention.

Example 1 A-beta Species Formation

A. 1 mg of human beta-amyloid 1-42 (H-5642, Bachem) and 500 uL of1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) (Aldrich) were chilled inseparate bottles on ice for 30 mins. Cold beta-amyloid 1-42 wassolubilized with cold HFIP. The mixture was incubated for 1 hr at roomtemperature until it turned clear. The resulting solution was then driedto a film under vacuum. The film was dissolved again in cold HFIP andincubated another 1 hr at room temperature. The resulting solution wasseparated into aliquots in several microcentrifuge tubes. HFIP wasremoved under vacuum, and the films were stored at −20° C. until use. Toprepare soluble oligomer, the film was dissolved in appropriate amountof dry DMSO (Sigma), and Ham's F12 media (Biosource) or PBS (Sigma,D8537) was added and incubated at 4° C. for 24 hours (finalconcentration of 200 uM or 0.9 mg/mL beta-amyloid in 2% DMSO). The tubecontaining the soluble oligomers was centrifuged at 13,000 rpm for 5mins, and its supernatant was transferred to a clean tube.

B. Insoluble A-beta (1-40) fibril formation: Lyophilized humanbeta-amyloid 1-40 peptide (Catalog number H-1194, Bachem) wassolubilized with HFIP to produce a dry clear film following the processdescribed above for beta-amyloid 1-42. The clear film of beta-amyloid1-40 was diluted with distilled water to achieve a 6 mg/mLconcentration. If the resulting solution had a cloudy appearance, it wasplaced in an ultrasonic bath until the solution became clear. The clearsolution was diluted to 1 mg/mL with PBS, pH 7.4 to obtain a finalconcentration of 1 mg/mL beta-amyloid 1-40, and then incubated at 37° C.on an orbital shaker at 200 RPM for 4 days. A cloudy solution wasproduced, and the fibril production was confirmed by measuring changesin fluorescence of Thioflavin T, as well as by atomic force microscopyand transmission electron microscopy. Fibrils were used immediatelyafter their production was confirmed.

Example 2 Benzofuran Derivative Preparation

A. Preparation of 2-bromo-3-bromomethyl benzofuran:2-bromo-3-bromomethyl benzofuran was prepared as described previously,using a modified procedure (Helv. Chim. Acta 1947, 30, 297). To asolution of 3-methylbenzofuran (4 g, 30.26 mmol) in carbon tetrachloride(20 ml) was added benzoyl peroxide (100 mg) and recrystallizedN-bromosuccinimide (NBS) (10.8 g, 2 equivalents). The mixture wasrefluxed for 3 hrs. The product formation was followed by GC-MS.Following analysis, 1.1 g NBS was added and the mixture was refluxed for1 hr. At this point, one more addition of NBS (1 g) followed by 1 hr ofreflux proved necessary. The solvent was stripped and replaced withethanol (1 2 ml), the mixture was cooled to −20 C, yielding a mass ofyellow crystals, which were filtered at −25 C The crystals of2-bromo-3-bromomethyl benzofuran were washed with ethanol 912 ml) at −40C, filtered and dried overnight (yield 7.672 g, 87%), better than 95%pure by GC-MS, which was immediately used in the next step. MS (m/e):291, 290, 289 (M⁺), 211, 209, 183,181, 146, 102, 75.

B. Preparation 2-bromo-3-hydroxymethyl benzofuran. 2-bromo-3-bromomethylbenzofuran from Step A (7.672 g, 26.45 mmol) was dissolved in dioxane(30 ml), followed by a solution of NaHCO3 (2.67 g, 1.2 eq.) in water (30ml). The mixture was refluxed for 1 hour while stirring vigorously,cooled to room temperature, diluted with water (150 ml) and extractedwith dichloromethane (5×). The extract was washed with brine, dried oversodium sulfate, and the solvent was removed under reduced pressure. Theresulting orange oil was dissolved in chloroform 912 ml and left tostand at −20 C. The resulting yellow prisms were filtered at −40C,washed with chloroform and filtered cold. Yield: 3.72 g (62%). MS (m/e):228, 226 (M⁺), 211, 209, 183, 181, 171, 169, 147, 118, 102, 91. ¹H-NMR(acetone-D₆): 4.29 (t,1H, J=6 Hz) 4.73 (d, 2H, J=6 Hz) 7.32 (m, 2H) 7.51(d,1H,J=8 Hz) 7.78 (dd, 1H, J=8 Hz, 2 Hz). ¹³C-NMR (acetone-D₆): 54.74,110.64, 119.96, 123.28, 124.57, 126.51, 128.12, 155.37.

C. Synthesis of 49b [2-(2-formyl-5-furanyl)-3-hydroxymethyl benzofuran].To the microwave vial was added the bromo-benzofuran derivative from B(0.1 mmol), the 2-formylfuran-5-boronic acid (Aldrich) (1.5 eq.),potassium carbonate (1.5 eq.), palladium dibenzylidene acetone (0.03eq.) and degassed dimethylacetamide (1 ml). The mixture was blanketedwith N₂ and heated in the microwave at 120 C for 10 minutes (initialpower 50 W). Water (2 ml) was added and the mixture was extracted withether (4×) and the crude extract was adsorbed on silica gel and purifiedby MPLC (hexanes/ethyl acetate gradient).MS (m/e): 242 (M⁺), 225, 213,196, 185, 168, 157, 139, 128, 102, 77. ¹H-NMR (acetone-D₆): 5.15 (s,2H), 7.19 (d, 1H, J=4 Hz), 7.35 (dd, 1H, J=8 Hz, 1 Hz), 7.44 (dd, 1H,J=8 Hz, 1 Hz), 7.56-7.65 (m, 2H), 7.93 (d, 1H, J=8 Hz), 9.76 (s,1H).¹³C-NMR (acetone-D₆).

D. Preparation of 2-bromo-3-formyl benzo[b]furan. To a 50 ml roundbottom flask were added powdered pyridinium chlorochromate (443 mg,2.055 mmol), Celite® (450 mg) and dry dichloromethane (20 ml). Asolution of the alcohol as in Formula I, wherein R¹═CH₂OH, R²═Br, X═O(226 mg, 1 mmol) in dichloromethane (1 ml) was added and the mixture wasstirred at r.t. in the dark for one hour. TLC (hexanes/ethyl acetate 4/1v/v) indicated complete conversion (R_(f) 0.7 vs. R_(f) 0.3 for startingmaterial). The mixture was diluted with ether, flushed through a 1 inplug of silicagel, the solvent was stripped and the residue was againflushed through a 1 in plug of silicagel, using hexanes/ethyl acetate2/1 v/v. to give the desired product as light orange crystals (205 mg,91%).

E. General procedure for the addition of organometallic reagents toaldehyde, as in Formula I, R¹═CHO, R²═Br, X═O: To a dry 5 ml flask wasadded the aldehyde (0.2-1 mmol), dry ether (1.5 ml) and the mixture wascooled to 0 C. The organometallic reagent solution (1.25 equivalents)was then added dropwise and the mixture was stirred at 0° C. for 30minutes. Then, GC-MS generally indicated complete conversion. The etherlayer was washed with aqueous citric acid, dried and the compound waspurified by MPLC using hexanes/ethylacetate gradient. When thecorresponding ketone was the desired product, the crude secondaryalcohol was oxidized according to the procedure outlined above.

F. General procedure for the reductive animation of 2-aryl-3-formylbenzofurans: To a 1 ml vial was added the aldehyde (0.05-0.1 mmol),dichloroethane (0.4 ml), 2 equivalents of the primary or secondaryamine, 1 equivalent of AcOH and 1.5 equivalents of NaBH(Oac)3. Themixture was stirred vigorously at room temperature. Most reactions werecomplete in 4-10 hrs as evidenced by GC-MS analysis. The solvent wasstripped with a nitrogen stream, ethyl acetate and silicagel (100-200mg) was added and the crude product was purified by MPLC using the solidsample technique.

G. General procedure for the preparation of 2-aryl-3-aminomethylbenzofurans: i) To a dry vial was added triphenylphosphine (275 mg, 1.05eq.) and phthalimide (149 mg, 1.02 eq.). The vial was stoppered andpurged with N2. Dry THF (2 ml) was added followed by a solution of thealcohol 2 (as in Formula I, R¹═CH₂OH, R²═Br, X═O) in THF (1 ml) andimmediately by diisopropyl-azo-dicarboxylate (DIAD), dropwise. Themixture warmed up as the phthalimide dissolved, and the vial was cooledwith an air stream. The resulting yellow solution was stirred at r.t.for 14 hrs. Silicagel was added to the reaction mixture and the solventwas stripped. The desired 2-bromo-3-(phthalimidomethyl)benzofuran wasobtained in 84% yield following purification by MPLC using hexanes ethylacetate 0-40% v/v gradient; ii) Introduction of various aryl moieties atthe 2 position, using the intermediate above, proceeded smoothlyfollowing the general Suzuki coupling methodology described above; iii)Deprotection of the 2-aryl-3-benzofuranylmethyl phthalimides thusobtained was achieved following treatment of the correspondingintermediate, as a 0.1M solution in isopropanol, with 1.05 equivalentsof N,N-dimethyl-1,3-propane diamine and microwave irradiation at 110 Cfor 10 minutes. Upon addition of silicagel the solvent was stripped offand the desired amine was obtained following MPLC using hexanes/ethylacetate containing 0.1% triethylamine.

Intermediates 34b, 35b (amides, ureas, urethanes in the compounds table)were prepared as follows: to a dry vial containing the amines (exampleNN1, c) (0.1 mmol in 1 ml dry dichloromethane) was added the isocyanate1.05 eq. and the mixture was stirred at room temperature for 4 hrs oruntil GC-MS analysis indicated complete conversion. The solvent wasstripped, the residue taken in 1 ml ethyl acetate and filtered through a3 ml SPE cartridge (silicagel) using ethyl acetate. The product was >95%pure (GC-MS) and was used without further purification. Similarly, acidchlorides were used for the synthesis of amides; in this case, 1 eq. oftriethylamine was used as an acid quencher. This procedure was also usedfor the synthesis of urethanes, when 1.25 eq. of chloroformate wasgenerally needed for the reaction to proceed to completion. Filtrationthrough an SPE cartridge of basic alumina produced compounds ofsufficient purity to be used without further MPLC purification.

Example 3 Radiolabeling of 49b

A. Preparation of [³H]2-bromo-3-hydroxymethyl benzofuran. To aldehyde2-bromo-3-formyl-benzofuran (15 mg, 66 μmol) in propan-2-ol:water 4:1(600 μl) was added a solution of NaBT₄ (5 Ci @ approx. 56 Ci/mmol) inpropan-2-ol:water 4:1 (600 μl). The solution was then stirred at roomtemperature for 2 hours. The residue was dissolved in ethyl acetate (5ml) and a sample was analyzed by silica TLC eluting indichloromethane:methanol (95:5). Yield: 15 Ci/mmol, 260 mCi (17 μmol).

B. Preparation of [³H]2-Br-3-acetoxymethyl benzofuran. Three equivalentsof acetic anhydride (5 μL) was added to 17 μmol of[³H]2-bromo-3-hydroxymethyl benzofuran, and the acetylation wasmonitored by TLC. After 2 hours, an additional 10 μl of acetic anhydridewas added and the mixture was swirled and left overnight. After a totalof 18 hours the reaction proceeded approximately 50%. A further 50 μl ofacetic anhydride was added and the mixture was left for a further 2hours. An additional 50 μl of acetic anhydride was added and thereaction mixture was left for a second night, after which the reactionappeared to have progressed to near completion.

The crude mixture was purified by HPLC using an Ultrasphere (BeckmanCoulter) ODS column eluting with a 0.1% TFA in water/acetonitrilegradient. The [³H]2-Br-3-acetoxymethyl benzofuran fractions were rotaryevaporated to dryness.

C. Preparation of [³H]49b from [³H]2-Br-3-acetoxymethyl benzofuran. To[³H]2-Br-3-acetoxymethyl benzofuran (100 mCi) was added K₂CO₃ (1.4 mg),5-formyl-furan-2-boronic acid (1.4 mg), Pd₂dba₃ (0.2 mg), and degasseddimethylacetamide (400 μl). The mixture was blanketed under nitrogen gasand heated with stirring at 80° C. for 6 hours. The reaction mixture wasanalyzed by TLC silica eluting in CH₂Cl₂:MeOH (95:5).

Deacetylation was performed by adding sodium hydroxide, 0.5 mg inTHF:methanol (1:1), to the mixture. The reaction was swirled and stirredat room temperature. Samples were periodically analyzed by TLC and after3 hours the reaction mixture was rotary evaporated to a lower volume andapplied to a 2 g Sep-Pak cartridge. The required fraction was countedand analyzed and purified by HPLC using an Ultrasphere C18 columneluting with a water/methanol gradient, followed by another purificationby HPLC using an Ultrasphere C18 column eluting with awater/acetonitrile gradient. The final product was analyzed by HPLC andmass spectrometry. Yield: specific activity of 13 Ci/mmol and 96.7%radiochemical purity.

Example 4 Binding Assays

The selectivity of 49b was demonstrated by two independent assays: an exvivo fluorescent binding assay and scintillation proximity assays(“SPA”). The use of an ex vivo assay with naïve rat brains had theadvantage of (1) decreasing confounding factors of target heterogeneitythat exists in transgenic animals, and it demonstrated the potentialusefulness of selectively binding probes such as 49b to (2) localizeoligomers in the context of brain tissue.

SPA employs direct binding of a radiolabeled probe, and demonstratedboth saturability and self-competition. We calculated a Kd of 50 μM forsoluble oligomers with no measurable affinity to fibrils (FIG. 1).Significantly, 49b shows no affinity against fibrils even at highconcentration, suggesting greater selectivity than reported antibodies.

For SPA and atomic force microscopy (AFM) experiments, preparations ofsoluble oligomers and fibrils contained 20% biotinylated beta-amyloid1-42 and 1-40, respectively (H-5642, H-5914, Bachem).

Example 5 Scintillation Proximity Assay

Lyophilized Streptavidin-Ysi beads (GE Healthcare) were reconstituted to100 mg/mL in deioniozed water, and then further diluted in deionizedwater to give a suspension containing 0.25 mg/10 μl water. Unlabeled 49band other benzofuran probes were dissolved in DMSO to give a final stockconcentration of 15 mM.

For the saturation-binding assay, isotopic dilution of radiolabeled 49bwith unlabeled 49b was performed. ³H-49b was added to 500 uM ofunlabeled 49b to a final concentration of 50 μCi/mL ³H-49b. The probesolution was then serially diluted in PBS to give six solutions giving6.6, 13.1, 26.3, 52.5, 105, or 210 uM in the final assay well. Eachconcentration of the probe solution was incubated with 12 μgbeta-amyloid 1-42 oligomer (20% biotin) or 1-40 (20% biotin) fibrils ina total volume of 90 μl. Assays were incubated at room temp for 2 hrsbefore addition of 10 μL YSi-streptavidin (0.25 mg) SPA bead. Assayswere set-up in triplicate in 96-well NBS plates (Costar). Assays wereincubated overnight and counted the following morning. Tritium-labeled49b was compared with tritium-labeled cimetide, caffeine, and AZT inbinding to soluble oligomers (FIG. 3). The specific activities of thefour probes were different, however the level of activity was the sameat the concentrations where the probes were tested. For this assay, 130nM of ³H-49b, 90 nM ³H-cimetidine, 14 nM ³H-caffeine, and 6 nM ³H-AZTwere tested for direct binding to 12 ug of beta-amyloid-42 oligomer (20%biotin). Assay conditions were the same as described in saturationbinding assay.

Tritium-labeled 49b was compared with two other tritium-labeledbenzofuran derivatives in binding to soluble oligomers. The specificactivities of ³H-49b, 37b, and 66b were similar to one another. For thisassay, 130 nM of ³H-49b, ³H-66b, ^(and 3)H-37b were tested for directbinding to 12 ug of beta-amyloid-42 oligomer (20% biotin). Assayconditions were the same as described in saturation binding assay.Representative data is shown in FIG. 4.

For the self-competition binding assay, assay wells contained 130 nM³H-49b and the indicated amount of unlabeled 49b (FIG. 2). Assayconditions were the same as described in saturation binding assay.

Data analyses were performed using Sigma Plot or GraphPad Prism® version4. Curves were fitted using non-linear regression. K_(D) values and IC₅₀values were estimated from the binding curves.

For the competition binding assay between tritium-labeled 49b andvarious unlabeled benzofuran analogs, assays contained 130 nM ³H-49b and50 uM of each of the unlabeled benzofuran analogs. As controls, 10 uMand 50 uM of unlabeled 49b were included in the assays. Representativedata is shown in Table 4 below.

TABLE 4 Compound % Inhibition No competitor control 0 (no inhibition)49b (10 microMolar) 29 49b (50 microMolar) 61  1c 24  2c 30  3c 38  4c41  5c 48  6c 51  7c 51  8c 51  9c 55 10c 57 11c 58 12c 59 13c 60 14c 6015c 68

A direct binding assay using tritium labeled 49b was performed againstoligomers or fibrils by scintillation proximity assay (SPA).Beta-amyloid containing 20% biotin and tritiated 49b were incubated insolution for two hours prior to addition of the Ysi-Streptavidin beads.While the binding reaction was done in solution, we wished to determineif beta-amyloid soluble oligomers and fibrils retained their respectiveconformation when bound to the SPA beads by AFM.

In the AFM studies, PVT-streptavidin SPA beads were used in place ofYsi-streptavidin beads because the smoother surface of the PVT beadsenhanced visibility of the bound beta-amyloid. AFM on PVT-streptavidinbeads demonstrated that the soluble oligomers and fibrils maintaineddistinct structure when bound to beads (FIG. 5B, 5E).

Saturation binding of 49b indicated a 50×10⁻⁶ mol/L binding constantwhen incubated with oligomers and a Bmax of 60-80 nmol/mg beta-amyloid,or a molar ratio of 1 probe: 3-4 beta-amyloid). No demonstrable affinitycould be measured when 49b was incubated with immobilized fibrils underidentical conditions (FIG. 1). Tritium labeled 49b binding to oligomerswas competed by unlabeled 49b with an IC₅₀ of 60 μM (FIG. 2). Tritiumlabeled non-benzofuran compounds (AZT, cimetidine, caffeine) did notbind to soluble oligomers indicating specificity of the benzofuran class(FIG. 3). Further, a set of substituted benzofuran analogues (Table 4)showed a wide range of response in competing with binding of tritiumlabeled 49b.

Example 6 AFM Imaging of A-beta Example 7 Ex vivo Assay Using Brains ofNaïve Rats

Fresh-frozen tissue sections of Sprauge-Dawley naïve rat brain (Taconic)10 microns in thickness were fixed with 10% formalin in PBS. Eachsection of the rat brain was then embedded in paraffin, an optional stepthat increases the shelf life of the sample.

Pre-formed soluble oligomers (100 μM), fibrils (100 μM), or monomerswere applied onto tissues, and the slides incubated in a humidifiedchamber for two hours at 37° C. The slides were washed with PBS threetimes, then incubated with blocking buffer consisting of 10% normal goatserum in 3% BSA in PBS. To verify the presence of beta-amyloid, 100 μLof a 1/250^(th) dilution of anti-beta-amyloid antibody, 6E10 (SignetLaboratories) was applied onto the tissues. The slides were incubatedfor 1.5 hours at room temperature or for 45 mins at 37° C. 1 mM stocksolutions of 49b and Thioflavin T were prepared in 50% ethanol. Theprobes were diluted to 50 μM in blocking buffer. 100 μL of each probewas added on to the slides containing 100 μL of 6E10. 6E10 antibody wasnot removed prior to addition of the probes. The slides were incubatedin a humid chamber for 1 hour at room temperature then washed threetimes with PBS.

A 1/100^(th) dilution of the secondary antibody Alexa Fluor 594-goatanti-mouse IgG (Molecular Probes) in PBS was applied onto the slides,and incubated at room temperature for 1.5 hours. Slides were washedthree times in PBS. Slides were coverslipped with AntiFade Gold(Molecular Probes) and incubated at least 4-6 hours before imaging.Microscopic examination and imaging was performed using a Leicawide-field fluorescence microscope using filter cube A (band pass340-380 nm with a 400 nm dichroic mirror and a long pass 400 nmsuppression filter) for 49b, cube E4 (band pass 436 nm with 455 dichroicmirror and a long pass 470 nm suppression filter) for Thioflavin T, andTX2 (band pass 520-600 nm with 595 nm dichroic mirror and 645/75 bandpass suppression filter) for 6E10 immunostaining.

TABLE 5 Long Pass Band Pass Band Pass Dichroic Suppression SuppressionCUBE (nm) Mirror (nm) Filter (nm) Filter Filter Cube A 340-380 400 400NA TX2 520-600 595 NA 645/75 E4 436 455 470 NA

Example 8 Histochemical Staining on the PDAPP Transgenic Mouse

3-month old and 24-month old PDAPP mouse brains were obtained from EliLilly (Indianapolis, Ind.). Fresh frozen 30 micron sections of thebrains were fixed in 10% formalin. For 49b, or Thioflavin S staining, 1mM stock solutions of 49b and Thioflavin S were prepared in 50% ethanol.The probes were diluted to 25 μM in 50% ethanol, and then applied ontothe PDAPP brain sections. The slides were incubated in a humid chamberat room temperature for 1 hr. The slides were washed three times withPBS and mounting medium (Molecular Probes) was added. The slides wereincubated at least 4-6 hours before imaging. For the carbonatepre-treatment of the brain sections, formalin-fixed sections wereincubated with 100 μL of carbonate buffer containing 0.025 M sodiumchloride and 0.1 M sodium carbonate for 45 mins at 37° C. The slideswere washed three times with PBS, then 49b or Thioflavin S were appliedon the slides as described above. Microscopic examination and imagingwas performed using a Leica wide-field fluorescence microscope usingfilter cube A for 49b and cube E4 for Thioflavin S. Co-staining of theprobes with 6E10 anti-beta-amyloid antibody in the PDAPP brain sectionswas described in the ex vivo assay using naïve rats.

Example 9 Ex Vivo Assay

Beta-amyloid fibrils and oligomers were produced from synthetic peptidefor binding assays and were demonstrated to have the expected distinctstructural features as shown by AFM (FIG. 5A, 5D). A directed library ofsubstituted benzofurans was synthesized based on a benzofuran seriesthat was described above to prevent the formation of higher orderfibrillar structuring of beta-amyloid. Most of the benzofuranssynthesized exhibited inherent fluorescence, so we designed a unique exvivo binding assay to test if the probes had the potential todifferentiate soluble oligomers from fibrils by fluorescence microscopy.Fresh frozen rat brains from naïve animals were incubated withpre-formed oligomers or fibrils on separate sections. Anti-beta-amyloidantibody immunoreactivity and Thioflavin T staining confirmed theimmobilization and localization of oligomers and fibrils (FIGS. 6A andC, top panel).

A preliminary screening using this ex vivo assay was performed. One ofthe probes that demonstrated robust differential binding between solubleoligomers and fibrils is the benzofuran 49b. This probe exhibited anexcitation peak at 350-370 nm and an emission maximum at 470 nm.Incubation of 49b on these sections resulted in clear staining ofoligomers with low background (FIG. 6B, bottom panel). Co-localizationof beta-amyloid staining and 49b confirmed this probe interacted withbeta-amyloid oligomers. No staining was observed when incubated onimmobilized fibrils (FIG. 6D, bottom panel). 49b did not stainbeta-amyloid 1-42 monomers or 1-40 monomers (data not shown).

Example 10 Binding in AD Animal Model

Brain sections from 24-month old PDAPP mice incubated with 49bdemonstrated a punctuate staining pattern that co-localized withbeta-amyloid immunoreactivity (FIGS. 7A and B). No staining was observedin young (3-month old) PDAPP mouse brains by either immunostaining by6E10 or 49b (FIG. 7C), confirming that 49b specifically binds solublebeta-amyloid.

Significantly, 49b staining was primarily found in the hippocampus andco-localized with Thioflavin S staining in the 24-month old PDAPP brainsections (FIG. 8). Since 49b and Thioflavin S have similar spectralproperties, co-localization was demonstrated by staining the sectionsfirst with 49b, washing the sections until the signal from 49b was notdetectable, then staining with Thioflavin S. To confirm that 49brecognized oligomers, PDAPP brain sections were pre-treated withcarbonate extraction buffer to remove soluble material. This buffer waspreviously used to remove pools of soluble beta-amyloid in the braintissues without removing beta-amyloid plaques 49b did not staincarbonate pre-treated brain sections, although Thioflavin S stainingremained (FIG. 9). Combined, these data support the conclusion that thein vivo selectivity of 49b and further indicates that beta-amyloidsoluble oligomers colocalize with fibrillar beta-amyloid in the PDAPPtransgenic mouse model.

Example 11 Fluorescence Titration Assays

Fresh solutions of 2-4 mM probe in methanol were appropriately dilutedwith PBS (pH 7.4) to obtain assay solutions with final concentrationrange of 0.1 nM to 100 μM probe in 300-400 μL PBS (pH 7.4, 2% methanol)with 10 μM A-beta (1-40) fibrils or 6.6 μM soluble A-beta (1-42). Aduplicate series of assay solutions were aliquoted with the volume ofsoluble A-beta or fibrils replaced by PBS. The set of solutions withprobe and soluble/fibrillar A-beta and the duplicate set without A-betawere prepared and the fluorescence spectra measured in triplicate.Spectral changes in the probe's fluorescence emission spectra that wereunique to the presence of A-beta indicated specific binding interactionsbetween the probe and A-beta species. This unique spectral changeallowed for the determination of the probe's binding affinity.Fluorescence emission spectra for binding affinity determinations aremeasured with fluorometric instrumentation known to those skilled in theart, including at least a spectrofluorometer and spectrograph.Equilibrium dissociation constants were calculated using a one-sitesaturation model of binding.

Select benzofuran congeners exhibited nanomolar binding affinity tosoluble A-beta (1-42) but no appreciable binding to A-beta (1-40)fibrils, as determined by fluorescence binding assays. A spectral changeof a probe's fluorescence that is unique to the presence of soluble orfibrillar A-beta was used to calculate the binding affinity of a probe.For example, the 22 nM K_(D) value (Table 6) indicates the selectivebinding of 37b (Table 2) for soluble A-beta beta with a characteristicincrease in fluorescence intensity (and quantum yield) approachingapproximately 5 times the fluorescence intensity (and quantum yield) of37b in 2% methanol in PBS. 37b in 10 μM of A-beta (1-40) fibrils hadminimal specific binding to the fibrils as demonstrated by an absence ofsignificant enhanced fluorescence intensity in the presence of fibrils.39b (Table 6) is an example of a benzofuran derivative that specificallybinds to A-beta (1-40) fibrils instead of soluble A-beta 42 suing theSPA assay. In contrast, 38b neither binds to A-beta in soluble norinsoluble fibrillar form.

TABLE 6 K_(D) of compound to K_(D) of compound to soluble A-betainsoluble A-beta Compound (1-42) (1-40) fibrils 38b Nonspecific bindingNonspecific binding 39b No binding <100 μM 1.68 μM 37b 22.8 nM Nobinding <100 μM

While the invention has been illustrated and described in typicalembodiments, it is not intended to be limited to the details shown,since various modifications and substitutions may be made withoutdeparting in any way from the spirit of the present invention. As such,further modifications and equivalents of the invention herein disclosedmay occur to persons skilled in the art using no more than routineexperimentation, and all such modifications and equivalents are believedto be within the spirit and scope of the invention as defined by thefollowing claims.

1. A compound of formula III

wherein X is oxygen, nitrogen, or sulfur; R¹ is substituted orunsubstituted alkyl, hydroxy, amide, urea, or urethane; and R³ ishalogen, formyl, C₁-C₃₂ substituted or unsubstituted, branched orstraight chain alkyl, cycloaliphatic, aryl, arylalkyl, or heteroaryl;and a label selected from radioisotopes, paramagnetic particles, andoptical particles.
 2. The compound of claim 1, wherein the label is aradioisotope selected from ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ¹²³I, ¹²⁵I,¹³¹I, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁹Fe, ⁷⁵Se, and ¹⁵²Eu.
 3. The compound of claim2, wherein the wherein the label is a radioisotope selected from ³H,¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, 123I, ¹²⁵I, ¹³¹I, ³⁶Cl, and ⁷⁵Se.
 4. Thecompound of claim 1, wherein the label is a paramagnetic particleselected from ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe.
 5. The compound ofclaim 1, wherein the label is an optical particle.
 6. A compoundaccording to claim 1, wherein R3 is selected from


7. A compound according to claim 1, wherein formula III is selected from


8. The compound of claim 7, wherein the label is a radioisotope selectedfrom ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ¹²³I, ¹²⁵I, ¹³¹I, ⁵¹Cr, ³⁶Cl, ⁵⁷Co,⁵⁹Fe, ⁷⁵Se, and ¹⁵²Eu.
 9. The compound of claim 7, wherein the whereinthe label is a radioisotope selected from ³H, ¹¹C, ¹⁴C, ¹⁸F, ³²P, ³⁵S,¹²³I, ¹²⁵I, ¹³¹I, ³⁶Cl, and ⁷⁵Se.
 10. The compound of claim 7, whereinthe label is a paramagnetic particle selected from ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy,⁵²Cr, and ⁵⁶Fe.
 11. The compound of claim 7, wherein the label is anoptical particle.